Adsorbent and process for preparing the same

ABSTRACT

An adsorbent for removing low and/or very low density lipoprotein from body fluid in extracorporeal circulation treatment, which comprises a water-insoluble porous hard gel with exclusion limit of 10 6  to 10 9  daltons on which a sulfated compound is immobilized by a covalent linkage.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of application Ser. No. 557,061 filed onDec. 1, 1983, now U.S. Pat. No. 4,576,928.

BACKGROUND OF THE INVENTION

The present invention relates to a novel adsorbent and a process forpreparing the same, more particularly, to an adsorbent for removingharmful substances to be removed from body fluid such as blood or plasmain extracorporeal circulation treatment.

There has been required a means for selectively removing harmfulsubstances which appear in body fluid and closely relate to a cause or aprogress of a disease. It is known that plasma lipoprotein, especiallyvery low density lipoprotein (hereinafter referred to as "VLDL") and/orlow density lipoprotein (hereinafter referred to as "LDL") contain alarge amount of cholesterol and cause arteriosclerosis. In hyperlipemiasuch as familial hyperlipemia or familial hypercholesterolemia, VLDLand/or LDL show several times higher values than those in normalcondition, and often cause arteriosclerosis such as coronaryarteriosclerosis. Although various types of treatments such as regimenand medications have been adopted, they have limitations in effect and afear of unfavorable side effects. Particularly in familialhypercholesterolemia, a plasma exchange therapy which is composed ofplasma removal and compensatory supplement of exogeneous human plasmaprotein solutions is probably the only treatment method being effectivenowadays. The plasma exchange therapy, however, has various defects suchas (1) a need for using expensive fresh plasma or plasma fractions, (2)a fear of infection by hepatitis viruses and the like, and (3) loss ofall plasma components containing not only harmful components but alsouseful ones, i.e. in case of lipoprotein, not only VLDL and/or LDL butalso high density lipoprotein (hereinafter referred to as "HDL") whichis an important factor to prevent Arteriosclerosis are lost. For thepurpose of solving the above defects, a selective removal of harmfulcomponents by a membrane and the like has been adopted. These methods,however, are insufficient in selectivity and cause a large loss ofuseful components from body fluid. There has been also tried a selectiveremoval of harmful components by means of adsorption. For example, asynthetic adsorbent such as active carbon or Amberlite XAD (a registeredtrademark, commercially available from Rohm & Hass Co.) has beenutilized for liver disease. Such an adsorbent however, has many defectssuch as poor selectivity and disability for removing high molecularweight compounds. Furthermore, for the purpose of increasingselectivity, there has been adopted an adsorbent based on the principleof affinity chromatography composed of a carrier on which a materialhaving an affinity for a substance to be specifically removed (suchmaterial is hereinafter referred to as "ligand") is immobilized. In thatcase, however, it is difficult to obtain a sufficient flow rate for anextracorporeal treatment because a carrier is a soft gel such asagarose. Accordingly, a particular modification in column shape isrequired in order to obtain a large flow rate and the risk of anoccasional clogging still remains. Therefore, a stable extracorporealcirculation cannot be achieved by the above method.

It is an object of the present invention to provide an adsorbent forselectively removing VLDL and/or LDL from body fluid such as blood orplasma in extracorporeal circulation treatment of hyperlipidemia.

A further object of the present invention is to provide a process forpreparing the adsorbent.

These and other objects of the present invention will become apparentfrom the description hereinafter.

SUMMARY OF THE INVENTION

In accordance with the present invention, there can be provided anadsorbent for removing LDL and/or VLDL from body fluid in extracorporealcirculation treatment comprising a water-insoluble porous hard gel withexclusion limit of 10⁶ to 10⁹ daltons on which a sulfated compound isimmobilized by a covalent linkage.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1 and 2 are graphs, respectively, showing relations between flowrate and pressure-drop obtained in Reference Examples 1 and 2, and

FIG. 3 is a chart of polyacrylamide disc gel electrophoresis obtained inExample 27.

DETAILED DESCRIPTION OF THE INVENTION

It is suitable that carriers used in the present invention have thefollowing properties:

(1) relatively high mechanical strength,

(2) low pressure-drop and no column clogging in case of passing bodyfluid through a column packed with a carrier,

(3) a large number of micro pores into which LDL and/or VLDL permeatessubstantially, and

(4) less change caused by a sterilizing procedure such as steamsterilization by autoclaving.

Therefore, the most suitable carrier used in the present invention is awater-insoluble porous polymer hard gel or a porous inorganic hard gel.

The porous hard gel used in the present invention is less swelled with asolvent and less deformed by pressure than a soft gel such as dextran,agarose or acrylamide.

The term "hard gel" and "soft gel" in the present invention is explainedas follows:

A hard gel is distinguished from a soft gel by the following methoddescribed in Reference Examples 1 and 2. That is, when a relationbetween flow rate and pressure-drop is determined by passing waterthrough a column uniformly packed with a gel, a hard gel shows a linearrelationship while a soft gel shows a non-linear relationship. In caseof a soft gel, a gel is deformed and consolidated over a certainpressure so that a flow rate does not increase further. In the presentinvention, a gel having the above linear relationship at least by 0.3kg/cm² is referred to as "hard gel".

A pore size of the porous hard gel is selected depending on molecularweight, shape, or size of a substance to be removed, and the mostsuitable pore size may be selected in each case. For measuring the poresize, there are various kinds of methods such as mercury porosimetry andobservation by an electron microscope as a direct measuring method. Withrespect to water-containing particles, however, the above methodssometimes cannot be applied. In such a case, an exclusion limit may beadopted as a measure of pore size. The term "exclusion limit" in thepresent invention means the minimum molecular weight of a molecularwhich cannot permeate into a pore in a gel permeation chromatography(cf. Hiroyuki Hatano and Toshihiko Hanai: Zikken Kosoku EkitaiChromatography (Experimental High-Pressure Liquid Chromatography),published by Kagaku Dojin). Phenomenally, a molecule having a molecularweight of more than exclusion limit is eluted near the void volume.Therefore, an exclusion limit can be determined by studying therelations between molecular weights and elution volumes using substancesof various molecular weights in a gel permeation chromatography. Anexclusion limit varies with a kind of substances to be excluded. In thepresent invention, an exclusion limit of the porous hard gel is measuredby using globular proteins and/or viruses, and the preferable exclusionlimit is 1×10⁶ to 1×10⁹. When the exclusion limit is more than 1×10⁹,the adsorbing amount of LDL and VLDL decreases with a decrease of amountof immobilized ligand and a decrease of surface area, and further amechanical strength of gel is reduced.

Removing VLDL and/or LDL being giant molecules having a molecular weightof more than 1×10⁶, a porous hard gel having an exclusion limit of lessthan 1×10⁶ is not practically available. On the other hand, a poroushard gel having an exclusion limit of from 1×10⁶ to several millionwhich is near a molecular weight of VLDL or LDL per se may bepractically available to a certain extent. A preferable exclusion limitfor removal of VLDL and/or LDL is 1×10⁶ to 1×10⁹, more preferably 1×10⁶to 1×10⁸.

With respect to a porous structure of the porous hard gel used in thepresent invention, a structure uniformly having pores at any part of thegel (hereinafter referred to as "uniform structure") is more preferablethan a structure having pores only on the surface of the gel. It ispreferred that a porosity of the gel is not less than 20%. The carriermay be selected from suitable shapes such as particle, fiber, sheet andhollow fiber. In case of using a carrier in the shape of particle,although a particle having a smaller size generally shows an excellentadsorbing capacity, the pressure-drop increases with an extremely smallsize. Therefore, a particle having a size of 1 μm to 5000 μm in diameteris preferred. Furthermore, it is preferred that a carrier has functionalgroups to be utilized for the immobilization of ligand or groups to beeasily activated. Examples of the group are, for instance, amino,carboxyl, hydroxyl, thiol, acid anhydride, succinylimide, chlorine,aldehyde, aminde, epoxy group, and the like.

Representative examples of the water-insoluble porous hard gel used inthe present invention are, for instance, a porous hard gel of asynthetic or a natural polymer such as stylene-divinylbenzene copolymer,cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linkedvinyl ether-maleic anhydride copolymer, cross-linked stylene-maleicanhydride copolymer or cross-linked polyamide, a porous cellulose gel,an inorganic porous hard gel such as silica gel, porous glass, porousalumina, porous silica alumina, porous hydroxyapatite, porous calciumsilicate, porous zirconia or porous zeolite, and the like. Of course itis to be understood that the porous hard gels used in the presentinvention are not limited to those set forth as examples above. Thesurface of the above-mentioned porous hard gel may be coated withpolysaccharides, synthetic polymers, and the like. These porous hardgels may be employed alone or in an admixture thereof.

In the above representative examples, some of the porous polymer hardgels composed of synthetic polymers have a fear of toxicity due tounreacted monomers and a less adsorbing capacity than that of a softgel.

Therefore, in the above representative examples, a porous cellulose gelis one of the particularly preferable carriers for the presentinvention, and it satisfies the above all four points required for thecarrier. In addition, the porous cellulose gel has various excellentadvantages such as hydrophilicity due to being composed of cellulose, alarge number of hydroxyl groups to be utilized for immobilization, lessnonspecific adsorption, and sufficient adsorbing capacity not inferiorto that of a soft gel due to its relatively high strength even with alarge porosity. Therefore, the porous cellulose gel on which a ligand isimmobilized provides a nearly ideal adsorbent.

As the porous cellulose gel used in the present invention, althoughcellulose per se is preferred, a cellulose derivative such as anesterified cellulose or an etherified cellulose, or a mixture ofcellulose and the cellulose derivatives may be employed. Examples of thecellulose derivative are, for instance, acetyl cellulose, methylcellulose, ethyl cellulose, carboxymethyl cellulose, and the like. It ispreferred that the cellulose gel is in the spherical shape. Thecellulose gel is prepared, for example, by dissolving or swellingcellulose and/or a cellulose derivatives with a solvent, dispersing theresulting mixture into another solvent being not admixed with the usedsolvent to make beads, and then regenerating the beads. The celluloseand/or cellulose derivatives may be cross-linked or not.

A porosity of a porous cellulose gel may be a measure of cellulosecontent. The cellulose content is expressed by the following formula:##EQU1## wherein W is dry gel weight (g), Vt is a volume of columnpacked with gel (ml) and Vo is a void volume (ml).

It is preferred that the cellulose content of the porous cellulose gelused in the present invention is 2% to 20%. In case of less than 2%, themechanical strength of gel is reduced, and in case of more than 20%, thepore volume is reduced.

Representative examples of the ligand used in the present invention areas follows:

As ligands to remove VLDL and/or LDL containing a large amount ofcholesterol and causing arteriosclerosis, sulfated compounds arepreferred as a ligand.

As the sulfated compound used for the ligand, there may be employed acompound obtained by sulfation of a hydroxy-containing compound.Examples of the sulfated compounds are, for instance, a sulfatedcarbohydrate, a sulfated polyhydric alcohol, polysulfated anethol, asulfated hydroxy-containing polymer such as polyvinyl alcohol orpolyhydroxyethyl methacrylate, and the like. Typical sulfatedcarbohydrate is a sulfated saccharide. Examples of the saccharides are,for instance a polysaccharide, a tetrose such as threose, a pentose suchas arabinose, xylose or ribose, a hexose such as glucose, galactose,mannose or fructose, a derivative thereof, a deoxysaccharide such asfucose, an amino-saccharide such as galactosamine, an acidic saccharidesuch as uronic acid, glucronic acid or ascorbic acid. Examples of thepolyhydric alcohol are, for instance, a glycol such as ethylene glycol,glycerol, sorbite pentaerythritol, and the like. Examples of thesulfated polysaccharides are heparin, dextran sulfate, chondroitinsulfate, chondroitin poly-sulfate, heparan sulfate, keratan sulfate,xylan sulfate, caronin sulfate, cellulose sulfate, chitin sulfate,chitosan sulfate, pectin sulfate, inulin sulfate, arginine sulfate,glycogen sulfate, polylactose sulfate, carrageenan sulfate, starchsulfate, polyglucose sulfate, laminarin sulfate, galactan sulfate, levansulfate and mepesulfate. Preferable examples of the above sulfatedcompounds are, for instance, sulfated polysaccharides such as heparin,dextran sulfate, chondroitin polysulfate, and/or the salts thereof, andparticularly preferable examples are a dextran sulfate and/or the saltthereof. Examples of the salt of the above sulfated compound are, forinstance, a water-soluble salt such as sodium salt or potassium salt,and the like.

Dextran sulfate and/or the salt thereof are explained in more detailhereinbelow.

Dextran sulfate and/or the salt thereof are sulfuric acid ester ofdextran being a polysaccharide produced by Leuconostoc mesenteroides,etc., and/or the salt thereof. It has been known that dextran sulfateand/or the salt thereof form a precipitate with lipoproteins in thepresence of a divalent cation, and dextran sulfate and/or the saltthereof having a molecular weight of about 5×10⁵ (intrinsic viscosity ofabout 0.20 dl/g) are generally employed for this precipitation. However,as shown in the following Example 39 of Run Nos. (1) and (2), a poroushard gel on which some of the above-mentioned dextran sulfate and/or thesalt thereof are immobilized is poor in affinity to VLDL and/or LDL. Asa result of extensive studies to solve the above problems, it has nowbeen found that dextran sulfate having an intrinsic viscosity of notmore than 0.12 dl/g, preferably not more than 0.08 dl/g, and a sulfurcontent of not less than 15% by weight has high affinity and selectivityto VLDL and/or LDL. Furthermore, the adsorbent of the present inventionemploying such dextran sulfate and/or the salt thereof as a ligand hashigh affinity and selectivity even in the absence of a divalent cation.Although a toxicity of dextran sulfate and/or the salt thereof is low,the toxicity increases with increasing of molecular weight. From thispoint of view, the use of dextran sulfate and/or the salt thereof havingan intrinsic viscosity of not more than 0.12 dl/g, preferably not morethan 0.08 dl/g can prevent a danger in case that the immobilized dextransulfate and/or the salt thereof should be released from a carrier. Inaddition, dextran sulfate and/or the salt thereof are less changed by asterilizing procedure such as steam sterilization by autoclaving,because they are linked mainly by α(1 6)-glycosidic linkage. Althoughthere are various methods for measuring a molecular weight of dextransulfate and/or the salt thereof, a method by measuring viscosity isgeneral. Dextran sulfate and/or the salt thereof, however, showdifferent viscosities depending on various conditions such as ionstrength, pH value, and sulfur content (content of sulfonic acid group).The term "intrinsic viscosity" used in the present invention means aviscosity of sodium salt of dextran sulfate measured in a neutral 1MNaCl aqueous solution, at 25° C. The dextran sulfate and/or the saltthereof used in the present invention may be in the form ofstraight-chain or branched-chain.

For coupling a ligand with a carrier, various methods such as physicaladsorption methods, ionic coupling methods and covalent coupling methodsmay be employed. In order to use the adsorbent of the present inventionin extracorporeal circulation treatment, it is important that the ligandis not released. Therefore, a covalent coupling method having a strongbond between ligand and carrier is preferred. In case of employing othermethods, a modification is necessary to prevent the release of ligand.If necessary, a spacer may be introduced between ligand and carrier.

It is preferred that a gel is activated by a reagent such as a cyanogenhalide, epichlorohydrin, a polyoxirane compound such as bisepoxide ortriazine halide, and then reacted with a ligand to give the desiredadsorbent. In that case, it is preferred that a gel having a group to beactivated such as hydroxyl group is employed as a carrier. In the abovereagents, epichlorohydrin or a polyoxirane compound such as bisepoxideis more preferred, because a ligand is strongly immobilized on a carrieractivated by using such a reagent and a release of a ligand is reduced.

Epichlorohydrin and a polyoxirane compound, however, show lowerreactivity, particularly lower to dextran sulfate and/or the saltthereof, because dextran sulfate and/or the salt thereof have hydroxylgroup alone as a functional group. Therefore, it is not easy to obtain asufficient amount of immobilized ligand.

As a result of extensive studies, it has now been found that thefollowing coupling method is preferred in case of using dextran sulfateand/or the salt thereof as a ligand. That is, a porous polymer hard gelis reacted with epichlorohydrin and/or a polyoxirane compound tointroduce epoxy groups into the gel, and then dextran sulfate and/or thesalt thereof is reacted with the resulting epoxy-activated gel in aconcentration of not less than 3% based on the weight of the wholereaction system excluding the dry weight of the gel, more preferably notless than 10%. This method gives a good immobilizing efficiency. In thatcase, a porous cellulose gel is particularly suitable as a carrier.

On the other hand, when a porous inorganic hard gel is employed as acarrier, it is preferred that the gel is activated with a reagent suchas an epoxysilane, e.g. γ-glycidoxypropyltrimethoxysilane or anaminosilane, e.g. γ-aminopropyltriethoxysilane, and then reacted with aligand to give the desired adsorbent.

The amount of immobilized ligand varies depending on properties of theligand used such as shape and activity. For sufficient removal of VLDLand/or LDL by using a polyanion compound, for instance, it is preferredthat the polyanion compound is immobilized in an amount of not less than0.02 mg/ml of an apparent column volume occupied by an adsorbent(hereinafter referred to as "bed volume"), economically 100 mg or less.The preferable range is 0.5 to 20 mg/ml of bed volume. Particularly, forremoval of VLDL and/or LDL by using dextran sulfate and/or the saltthereof as a ligand, it is preferred that the amount of immobilizedligand is not less than 0.2 mg/ml of bed volume. After the couplingreaction, the unreacted polyanion compound may be recovered for reuse bypurification, etc.

It is preferred that the remaining unreacted active groups are blockedby ethanolamine, and the like.

In accordance with the present invention, an adsorbent composed ofporous cellulose gel having an exclusion limit of 10⁶ to 10⁸ and aparticle size of 30 to 200 μm on which sodium salt of dextran sulfatehaving an intrinsic viscosity of not more than 0.12 dl/g and a sulfurcontent of not less than 15% by weight is immobilized, is particularlysuitable for removal of VLDL and/or LDL in extracorporeal circulationtreatment of hypercholesterolemia.

The adsorbent of the present invention may be employed for various kindsof use. Representative example of the use is extracorporeal circulationtreatment performed by incorporating a column into extracorporealcirculation circuit and passing body fluid such as blood or plasmathrough the column, the column being packed with the adsorbent of thepresent invention. The use of the adsorbent is not necessarily limitedto the above example.

The adsorbent of the present invention can be subjected to steamsterilization by autoclaving so long as the ligand is not largelydegenerated, and this sterilization procedure does not affect on micropore structure, particle shape and gel volume of the adsorbent.

The present invention is more specifically described and explained bymeans of the following Reference Examples and Examples, and it is to beunderstood that the present invention is not limited to the ReferenceExamples and Examples.

REFERENCE EXAMPLE 1

Biogel A5m (a commercially available agarose gel made by Biorad Co.,particle size: 50 to 100 mesh) as a soft gel and Toyopearl HW65 (acommercially available cross-linked polyacrylate gel made by Toyo SodaManufacturing Co., Ltd., particle size: 50 to 100 μm) and CellulofineGC-700 (a commercially available porous cellulose gel made by ChissoCorporation, particle size: 45 to 105 μm) as a hard gel were uniformlypacked, respectively, in a glass column (inner diameter: 9 mm, height:150 mm) having filters (pore size: 15 μm) at both top and bottom of thecolumn. Water was passed through the thus obtained column, and arelation between flow rate and pressure-drop was determined. The resultsare shown in FIG. 1. As shown in FIG. 1, flow rate increasedapproximately in proportion to increase of pressure-drop in the porouspolymer hard gels. On the other hand, the agarose gel was consolidated.As a result, increasing pressure did not make flow rate increase.

REFERENCE EXAMPLE 2

The procedures of Reference Example 1 were repeated except that FPG 2000(a commercially available porous glass made by Wako Pure ChemicalIndustry Ltd., particle size: 80 to 120 mesh) instead of porous polymerhard gels was employed as a porous inorganic hard gel. The results areshown in FIG. 2. As shown in FIG. 2, flow rate increased approximatelyin proportion to increase of pressure-drop in the porous glass, whilenot in the agarose gel.

EXAMPLE 1

Toyopearl HW55 (a commercially available cross-linked polyacrylate gelmade by Toyo Soda Manufacturing Co., Ltd., exclusion limit: 7×10⁵,particle size: 50 to 100 μm) having a uniform structure was employed asa carrier.

To 10 ml of the gel were added 6 ml of saturated NaOH aqueous solutionand 15 ml of epichlorohydrin, and the reaction mixture was subjected toreaction with stirring at 50° C. for 2 hours. The gel was washedsuccessively with alcohol and water to introduce epoxy groups into thegel. To the resulting epoxy-activated gel was added 20 ml ofconcentrated aqueous ammonia, and the reaction mixture was stirred at50° C. for 2 hours to introduce amino groups into the gel.

Three ml portion of the thus obtained activated-gel containing aminogroups was added to 10 ml of aqueous solution (pH 4.5) containing 200 mgof heparin. To the resulting reaction mixture was added 200 mg of1-ethyl-3-(dimethylaminopropyl)-carbodiimide while maintaining thereaction mixture at pH 4.5, and then the reaction mixture was shaken at4° C. for 24 hours. After completion of the reaction, the resultingreaction mixture was washed successively with 2M NaCl aqueous solution,0.5M NaCl aqueous solution and water to give the desired gel on whichheparin was immobilized (hereinafter referred to as "heparin-gel"). Theamount of immobilized heparin was 2.2 mg/ml of bed volume.

EXAMPLES 2 TO 4

The procedures of Example 1 were repeated except that Toyopearl HW60(exclusion limit: 1×10⁶, particle size: 50 to 100 μm), Toyopearl HW 65(exclusion limit: 5×10⁶, particle size: 50 to 100 μm) and Toyopearl HW75(exclusion limit: 5×10⁷, particle size: 50 to 100 μm) instead ofToyopearl HW55 were employed, respectively, to give each heparin-gel.Toyopearl HW60, Toyopearl HW65 and Toyopearl HW75 are all commerciallyavailable cross-linked polyacrylate gels having a uniform structure madeby Toyo Soda Manufacturing Co., Ltd. The amounts of immobilized heparinwere, respectively, 1.8 mg, 1.4 mg and 0.8 mg/ml of bed volume.

EXAMPLE 5

Cellulofine GC 700 (a commercially available porous cellulose gel madeby Chisso Corporation, exclusion limit: 4×10⁵, particle size: 45 to 105μm) having a uniform structure was employed as a carrier.

The gel was filtered with suction, and 4 g of 20% NaOH and 12 g ofheptane were added to 10 g of the suction-filtered gel. One drop ofTween 20 (nonionic surfactant) was further added to the reaction mixturewhich was stirred for dispersing the gel. After stirring at 40° C. for 2hours, 5 g of epichlorohydrin was added to the reaction mixture whichwas further stirred at 40° C. for 2 hours. After the reaction mixturewas allowed to stand, the resulting supernatant was discarded, and thegel was washed with water to introduce epoxy groups into the gel. To theresulting epoxy-activated gel was added 15 ml of concentratedaqueousammonia, and the reaction mixture was stirred at 40° C. for 1.5 hours,filtered with suction and washed with water to introduce amino groupsinto the gel.

Three ml portion of the thus obtained activated gel containing aminogroups was added to 10 ml of aqueous solution (pH 4.5) containing 200 mgof heparin. To the resulting reaction mixture was added 200 mg of1-ethyl-3-(dimethylaminopropyl)-carbodiimide while maintaining thereaction mixture at pH 4.5, and then the reaction mixture was shaken at4° C. for 24 hours. After completion of the reaction, the resultingreaction mixture was washed successively with 2M NaCl aqueous solution,0.5M NaCl aqueous solution and water to give the desiredheparin-Cellulofine A-3. The amount of immobilized heparin was 2.5 mg/mlof bed volume.

EXAMPLES 6 TO 7

The procedures of Example 5 were repeated except that Cellulofine A-2(exclusion limit: 7×10⁵, particle size: 45 to 105 μm) and CellulofineA-3 (exclusion limit: 5×10⁷, particle size: 45 to 105 μm) instead ofCellulofine GC 700 were employed, respectively, to give eachheparin-gel. Both Cellulofine A-2 and Cellulofine A-3 are commerciallyavailable porous cellulose gels having a uniform structure made byChisso Corporation. The amounts of immobilized heparin were,respectively, 2.2 mg and 1.8 mg/ml of bed volume.

EXAMPLE 8

The procedures of Example 5 were repeated except that Cellulofine A-3having a particle size of 150 to 200 μm instead of 45 to 105 μm wasemployed. The amount of immobilized heparin was 1.5 mg/ml of bed volume.

EXAMPLE 9

The procedures of Example 1 were repeated except that Toyopearl HW65instead of Toyopearl HW55 and chondroitin polysulfate instead of heparinwere employed, to give the desired chondroitin polysulfate-ToyopearlHW65. The amount of immobilized chondroitin polysulfate was 1.2 mg/ml ofbed volume.

EXAMPLE 10

To 4 ml of Cellulofine A-3 was added water to make the volume up to 10ml, and then 0.5 mole of NaIO₄ was added. After stirring at a roomtemperature for one hour, the reaction mixture was washed with water byfiltration to introduce aldehyde groups into the gel. The thus obtainedgel was suspended in 10 ml of phosphate buffer of pH 8 and stirred at aroom temperature for 20 hours after addition of 50 mg ofethylenediamine. The gel was filtered off and then suspended in 10 ml of1% NaBH₄ solution. After reducing reaction for 15 minutes, the reactionmixture was filtered and washed with water to introduce amino groupsinto the gel.

In 10 ml of 0.25M NaIO₄ solution was dissolved 300 mg of sodium salt ofdextran sulfate. After stirring at a room temperature for 4 hours, 200mg of ethylene glycol was added to the resulting solution and stirredfor one hour. The resulting solution was adjusted to pH 8, and then theabove gel containing amino groups was suspended in the solution andstirred for 24 hours. After completion of the reaction, the gel wasfiltered, washed with water, and then suspended in 10 ml of 1% NaBH₄solution. The resulting suspension was subjected to reducing reactionfor 15 minutes and washed with water by filtration to give the desiredsodium salt of dextran sulfate-Cellulofine A-3. The amount ofimmobilized sodium salt of dextran sulfate was 0.5 mg/ml of bed volume.

EXAMPLE 11

Cellulofine A-3 was treated in the same manner as in Example 5 tointroduce epoxy groups into the gel.

Two ml of the thus obtained epoxy-activated gel was added to 2 ml ofaqueous solution containing 0.5 g of sodium salt of dextran sulfate(intrinsic viscosity 0.055 dl/g, average polymerization degree: 40,sulfur content: 19% by weight), and the reaction mixture was adjusted topH 12. The concentration of sodium salt of dextran sulfate was about 10%by weight. The resulting reaction mixture was filtered and washedsuccessively with 2M NaCl aqueous solution, 0.5M NaCl aqueous solutionand water to give the desired sodium salt of dextran sulfate-CellulofineA-3. The remaining unreacted epoxy groups were blocked withmonoethanolamine. The amount of immobilized sodium salt of dextransulfate was 1.5 mg/ml of bed volume.

EXAMPLE 12

To 5 g of suction-filtered Cellulofine A-3 were added 2.5 ml of1,4-butanediol diglycidyl ether and 7.5 ml of 0.1N NaOH aqueoussolution, and the reaction mixture was stirred at a room temperature for18 hours to introduce epoxy groups into the gel.

The thus obtained epoxy-activated gel was reacted with sodium salt ofdextran sulfate in the same manner as in Example 11 to give the desiredsodium salt of dextran sulfate-Cellulofine A-3. The amount ofimmobilized sodium salt of dextran sulfate was 1.8 mg/ml of bed volume.

EXAMPLE 13

The procedures of Example 11 were repeated except that Cellulofine A-6(a commercially available porous cellulose gel made by ChissoCorporation, exclusion limit: 1×10⁸, particle size: 45 to 105 μm) havinga uniform structure instead of Cellulofine A-3 was employed to give thedesired sodium salt of dextran sulfate-Cellulofine A-6. The amount ofimmobilized sodium salt of dextran sulfate was 1.2 mg/ml of bed volume.

EXAMPLE 14

Toyopearl HW65 was treated in the same manner as in Example 1 tointroduce epoxy groups into the gel.

Two ml of the thus obtained epoxy-activated gel was treated in the samemanner as in Example 11 to give the desired sodium salt of dextransulfate-Toyopearl HW65. The amount of immobilized sodium salt of dextransulfate was 0.4 mg/ml of bed volume.

EXAMPLE 15

FPG 2000 (exclusion limit: 1×10⁹, particle size: 80 to 120 mesh, averagepore size: 1950 Å) was heated in diluted nitric acid for 3 hours. Afterwashing and drying, the gel was heated at 500° C. for 3 hours and thenrefluxed in 10% γ-aminopropyltriethoxysilane solution in toluene for 3hours. After washing with methanol, a γ-aminopropyl-activated glass wasobtained.

Two g of the thus obtained activated glass was added to 10 ml of aqueoussolution (pH 4.5) containing 200 mg of heparin. The reaction mixture wastreated in the same manner as in Example 1 to give the desiredheparin-FPG 2000. The amount of immobilized heparin was 1.2 mg/ml of bedvolume.

EXAMPLES 16 TO 18

The procedures of Example 15 were repeated except that FPG 700 (acommercially available porous glass made by Wako Pure Chemical IndustryLtd., exclusion limit: 5×10⁷, particle size: 80 to 120 mesh, averagepore size: 70 Å), FPG 1000 (a commercially available porous glass madeby Wako Pure Chemical Industry Ltd., exclusion limit: 1×10⁸, particlesize: 80 to 120 mesh, average pore size: 1091 Å) and Lichrospher Si4000(a commercially available porous silica gel made by Merck & Co. Inc.,exclusion limit: 1×10⁹, average particle size: 10 μm, average pore size:4000 Å) instead of FPG 2000 were employed. The amounts of immobilizedheparin were, respectively, 3.2 mg, 2.2 mg and 0.5 mg/ml of bed volume.

EXAMPLE 19

The procedures of Example 15 were repeated except that chondroitinpolysulfate instead of heparin was employed to give the desiredchondroitin polysulfate-FPG 2000. The amount of immobilized chondroitinpolysulfate was 1.0 mg/ml of bed volume.

EXAMPLE 20

FPG 2000 was treated in the same manner as in Example 15 to introduceγ-aminopropyl groups into the gel. The thus obtained activated gel wasreacted with sodium salt of dextran sulfate in the same manner as inExample 10 to give the desired sodium salt of dextran sulfate-FPG 2000.The amount of immobilized sodium salt of dextran sulfate was 0.5 mg/mlof bed volume.

EXAMPLE 21

FPG 2000 was refluxed in 10% solution ofγ-glycidoxypropyltrimethoxysilane for 3 hours and then washed withmethanol. The thus obtained activated gel was reacted with sodium saltof dextran sulfate in the same manner as in Example 11 except that thereaction was carried out at pH 8.5 to 9 and at 45° C. to give thedesired sodium salt of dextran sulfate-FPG 2000.

EXAMPLE 22

The procedures of Example 11 were repeated except that sodium salt ofglucose sulfate instead of dextran sulfate was employed to give thedesired sodium salt of glucose sulfate-Cellulofine A-3.

The amount of immobilized sodium salt of glucose was 1.0 mg/ml of bedvolume.

EXAMPLE 23

The procedures of Example 11 were repeated except that sodium salt ofpolyvinyl alcohol sulfate instead of dextran sulfate was employed togive the desired sodium salt of polyvinyl alcohol sulfate-CellulofineA-3.

The amount of immobilized sodium salt of polyvinyl alcohol sulfate was1.5 mg/ml of bed volume.

TEST EXAMPLE 1

Each adsorbent obtained in Examples 1 to 23 was uniformly packed in acolumn (internal volume: about 3 ml, inner diameter: 9 mm, height: 47mm) and 18 ml of plasma containing 200 U of heparin was passed throughthe column at a flow rate of 0.3 ml/minute with varying the plasmaorigins depending on the kind of the desired substance to be removed.That is, human plasma derived from familial hypercholesterolemia, normalhuman plasma, normal human plasma containing about 100 μg/ml of acommercially available endotoxin, human plasma derived from rheumatism,human plasma derived from systemic lupus erythematosus and human plasmaderived from myasthenia gravis were used, respectively, for the tests ofremoving VLDL and/or LDL; IgG, C_(1q) or haptoglobin; endotoxin;rheumatoid factor; anti-DNA antibody or DNA; and anti-acetylcholinereceptor antibody. The pressure-drop in the column was 15 mmHg or lessthroughout the test period and no crogging was observed. In eachadsorbent, LDL, VLDL, HDL, total protein in plasma which was passedthrough the column was determined to obtain a removal efficiency. Theresults are summarized in Table 1.

                                      TABLE 1    __________________________________________________________________________    Example               Coupling Removal rate (%)    No.  Ligand  Carrier  method   LDL + VLDL                                           HDL Protein*.sup.(1)    __________________________________________________________________________     1   Heparin Toyopearl HW55                          Epichlorhydrin-                                   23      5   2                          ammonia     2   Heparin Toyopearl HW60                          Epichlorhydrin-                                   31      11  3                          ammonia     3   Heparin Toyopearl HW65                          Epichlorhydrin-                                   54      12  3                          ammonia     4   Heparin Toyopearl HW75                          Epichlorhydrin-                                   51      8   4                          ammonia     5   Heparin Cellulofine                          Epichlorhydrin-                                   15      0   3                 GC-700   ammonia     6   Heparin Cellulofine A-2                          Epichlorhydrin-                                   26      2   2                          ammonia     7   Heparin Cellulofine A-3                          Epichlorhydrin-                                   56      2   3                 (particle size:                          ammonia                 45 to 105 μm)     8   Heparin Cellulofine A-3                          Epichlorhydrin-                                   55      3   2                 (particle size:                          ammonia                 150 to 200 μm)    15   Heparin FPG 2000 amminosilane                                   57      13  8    16   Heparin FPG 700  amminosilane                                   16      8   7    17   Heparin FPG 1000 amminosilane                                   28      9   9    18   Heparin Lichrosphere                          amminosilane                                   24      5   14                 Si 4000     9   Chondroitin                 Toyopearl HW65                          Epichlorhydrin-                                   46      12  9         polysulfate      ammonia    19   Chondroitin                 FPG 2000 aminosilane                                   45      15  13         polysulfate    22   glucose Cellulofine A-3                          Epichlorhydrin                                   38      5   2         sulfate    23   polyvinyl                 Cellulofine A-3                          Epichlorhydrin                                   47      3   2         alcohol         sulfate    10   sodium salt of                 Cellulofine A-3                          NaIO.sub.4 --diamine                                   38      2   2         dextran sulfate    20   sodium salt of                 FPG 2000 NaIO.sub.4 --diamine                                   37      12  4         dextran sulfate    11   sodium salt of                 Cellulofine A-3                          Epichlorhydrin                                   50      3   1         dextran sulfate    12   sodium salt of                 Cellulofine A-3                          Bisepoxide                                   65      3   2         dextran sulfate    13   sodium salt of                 Cellulofine A-6                          Epichlorhydrin                                   60      3   2         dextran sulfate    14   sodium salt of                 Toyopearl HW65                          Epichlorhydrin                                   42      5   5         dextran sulfate    21   sodium salt of                 FPG 2000 Epoxysilane                                   60      8   11         dextran sulfate    __________________________________________________________________________     *.sup.(1) Protein other than lipoprotein, i.e. total protein  lipoprotein

EXAMPLE 24 [Effects of intrinsic viscosity and sulfur content of dextransulfate and/or the salt thereof]

Cellulofine A-3 was treated in the same manner as in Example 5 tointroduce epoxy groups into the gel. The thus obtained epoxy-activatedgel was reacted with each sodium salt of dextran sulfate having theintrinsic viscosity and sulfur content shown in the following Table 2(Run Nos. (1) to (7)) in the same manner as in Example 11.

One ml portion of the resulting each adsorbent was packed in a column,and then 6 ml of human plasma containing 300 mg/dl of total cholesterolderived from a familial hypercholesterolemia patient was passed throughthe column at a flow rate of 0.3 ml/minute. The removal efficiency forLDL was determined from the amount of adsorbed LDL measured by using thetotal amount of cholesterol as an indication. That is, the amount ofcholesterol in the human plasma used was mostly derived from LDL. Theresults are shown in Table 2.

                                      TABLE 2    __________________________________________________________________________                    Concentration of                               Amount of                    sodium salt of                               immobilized       Intrinsic            Sulfur  dextran sulfate                               sodium salt of                                           Removal    Run       viscosity            content in the reaction system                               dextran sulfate                                           efficiency    No.       (dl/g)            (% by weight)                    (% by weight)                               (mg/ml of bed volume)                                           (%)    __________________________________________________________________________    (1)       0.20 17.7    about 10   4.2         18    (2)       0.124             5.7    "          2.5         17    (3)       0.027            17.7    "          2           62    (4)       0.055            19.0    "          1.5         50    (5)       0.083            19.2    "          4.0         44    (6)       0.118            17.7    "          4.3         39    (7)       0.055            19.0    2.5        0.15        32    __________________________________________________________________________

EXAMPLE 25 [Effect of amount of epoxy group introduced]

Toyopearl 65 was treated in the same manner as in Example 1 to introduceepoxy groups into the gel and CSKA-3 (a cmmercially available porouscellulose gel made by Chisso Corporation, exclusion limit: 5×10⁷,particle size: 45 to 105 μm) having a uniform structure was treated inthe same manner as in Example 5 to introduce epoxy groups into the gel.The amounts of epoxy groups introduced were, respectively, 250 μmolesand 30 μmoles/ml of bed volume.

Each gel was reacted with sodium salt of dextran sulfate (intrinsicviscosity: 0.027 dl/g, sulfur content: 17.7% by weight) in the samemanner as in Example 11 except that the concentration of sodium salt ofdextran sulfate based on the weight of the whole reaction systemexcludig the dry weight of the gel was charged.

The thus obtained adsorbent was subjected to the determination ofremoval efficiency for LDL in the same manner as in Example 24. Theresults are summarized in Table 3.

                                      TABLE 3    __________________________________________________________________________             Amount of epoxy                      Amount of immobilized sodium                                     Concentration             group introduced                      salt of dextran sulfate                                     of sodium salt                                             Removal             (μmole/ml of                      mg/ml of                             μg/μmole of                                     of sulfate                                             efficiency    Carrier  bed volume)                      bed volume                             epoxy group                                     (% by weight)                                             (%)    __________________________________________________________________________    Toyopearl HW65             250      0.4    1.6     13      40    CSKA-3   30       0.15   5       2.5     36    "        30       2.3    76      13      63    __________________________________________________________________________

EXAMPLE 26

One ml portion of the adsorbent obtained in Example 24 of Run No. (3)was uniformly packed in a column having an internal volume of 1 ml, and6 ml of normal human plasma containing LDL and HDL cholesterol in theratio of approximately 1:1 was passed through the column. LDL in theplasma passed through the column was greatly reduced, while HDL wasscarcely reduced.

EXAMPLE 27

One ml portion of the adsorbent obtained in Example 24 of Run No. (3)was uniformly packed in a column having an internal volume of 1 ml, and6 ml of normal rabbit plasma containing lipoproteins of VLDL, LDL andHDL was passed through the column. The plasma obtained before and afterthe column treatment were, respectively, examined by polyacrylamide discgel electrophoresis. The results are shown in FIG. 3. In FIG. 3, curvesA and B show, respectively, the results obtained before and after thecolumn treatment. The axis of ordinates indicates the absorbance at 570nm and the axis of abscissas indicates the migration positions at whichbands of VLDL, LDL and HDL were, respectively appeared.

As shown in FIG. 3, VLDL and LDL were significantly adsorbed, while HDLwas not.

EXAMPLE 28

The adsorbents obtained in Examples 1 to 7 and 11 to 14 were sterilizedin an autoclave at 120° C. for 15 minutes. Each resulting sterilizedadsorbent was subjected to the determination of removal efficiency forLDL in the same manner as in Test Example 1. As a result, the removalefficiencies were not inferior to those obtained without sterilizing byautoclaving. In addition, pressure-drop was not changed.

What we claim is:
 1. An adsorbent for removing low and/or very lowdensity lipoprotein from body fluid in extracorporeal circulationtreatment, which comprises a water-insoluble porous hard gel withexclusion limit of 10⁶ to 10⁹ daltons on which a sulfated compound isimmobilized by a covalent linkage; said sulfated compound being acompound obtained by sulfation of a hydroxy-containing compound.
 2. Theadsorbent of claim 1, wherein said water-insoluble porous hard gel is awater-insoluble porous polymer hard gel.
 3. The adsorbent of claim 2,wherein said water-insoluble porous polymer hard gel is a porouscellulose gel.
 4. The adsorbent of claim 1, wherein said water-insolubleporous hard gel is a porous inorganic hard gel.
 5. The adsorbent ofclaim 4, wherein said water-insoluble inorganic hard gel is a memberselected from the group consisting of porous glass, porous silica geland porous alumina.
 6. The adsorbent of claim 1, wherein the sulfatedcompound is a sulfated carbohydrate.
 7. The adsorbent of claim 6,wherein the sulfated carbohydrate is a sulfated saccharide.
 8. Theadsorbent of claim 7, wherein the sulfated saccharide is a sulfatedpolysaccharide.
 9. The adsorbent of claim 8, wherein the sulfatedpolysaccharide is a member selected from the group consisting ofheparin, dextran sulfate, condroitin sulfate and salts thereof.
 10. Theadsorbent of claim 9, wherein the dextran sulfate, a salt thereof or amixture of the dextran sulfate and the salt has an intrinsic viscosityof not more than 0.12 dl/g and a sulfer content of not less than 15% byweight.
 11. The adsorbent of claim 1, wherein the sulfated compound is asulfated polyhydric alcohol.
 12. The adsorbent of claim 1, wherein theexclusion limit is 10⁶ to 10⁸ daltons.
 13. The adsorbent of claim 1,wherein said sulfated compound is immobilized in an amount of 0.02 to100 mg/ml of bed volume.
 14. The adsorbent of claim 13, wherein thesulfated compound is immobilized in an amount of not less than 0.2 mg/mlof bed volume.
 15. A process of preparing an adsorbent for removing lowand/or very low density lipoprotein from body fluid in extracorporealcirculation treatment comprising a water-insoluble porous hard gel withexclusion limit of 10⁶ to 10⁹ daltons on which a sulfated compound isimmobilized, wherein said water-insoluble porous hard gel is reactedwith epichlorhydrin or a polyoxirane compound to introduce epoxy groupson to the gel, and then the resulting epoxy-activated gel is reactedwith the sulfated compound; said sulfated compound being a compoundobtained by sulfation of a hydroxy-containing compound.
 16. The processof claim 15, wherein said water-insoluble hard gel is a water-insolubleporous polymer hard gel.
 17. The process of claim 16, wherein saidwater-insoluble porous polymer hard gel is a porous cellulose gel. 18.The process of claim 15, wherein said sulfated compound is dextransulfate, a salt thereof or a mixture of the dextran sulfate and thesalt; said dextran sulfate, the salt thereof or the mixture of thedextran sulfate and the salt being reacted with the epoxy-activated gelin a concentration of not less than 3% by weight based on the weight ofthe whole reaction system excluding the dry weight of the porous hardgel.
 19. The process of claim 18, wherein the porous hard gel is aporous cellulose gel.